1.Orthopoxvirus pathogenesis A goal of EVPS is to understand virus-host interactions and exploit them for countermeasure development. Previously, we had established that infection of rhesus macaques with cowpox virus (CPXV) resulted in a disease that resembled human hemorrhagic smallpox, a rare and nearly 100% lethal disease associated with secondary bacterial infections. We compared the impact of antibiotic treatment on disease progression. 8 NHPs were IV inoculated with CPXV, 4 were treated with antibiotics, 4 were not. We did not observe a difference in onset of clinical signs or outcome between the groups. We did observe some differences in clinical parameters such as complete blood count and complete metabolic panel serum chemistry values. As an additional step in development of the IV NHP CPXV model we performed a serial euthanasia study with cardiac MR to evaluate cardiac function through the course of disease. We observed acute changes in heart function that coincided with subjects reaching moribund endpoint criteria. We also altered the route of inoculation of CPXV in macaques to determine if small particle aerosol results in disease that more closely resembles human smallpox. We observed an LD100 and LD50 and disease progression was different than IV inoculation and did not result in hemorrhagic signs or secondary bacterial infections. We also performed chest CT to evaluate lung progression for further development of medical imaging techniques for use in infectious disease studies. To further develop our CPXV model as a surrogate for smallpox, large particle aerosol inoculation was performed. In contrast to the small particle aerosol inoculation experiments, large particle aerosol inoculation resulted in reduced inflammation in the lung as evaluated by CT. With large particle aerosol inoculation, we were unable to achieve an LD100, even at 100 fold more virus than small particle aerosol. Our studies indicate the route of inoculation influences disease outcome. Our third project studying orthopox pathogenesis is based on our Backwards Matched Longitudinal Analysis which identified several cytokines that were statistically associated with lethal or non-lethal outcome in NHPs. The duration and intensity of certain cytokines was associated with survival (IFN-gamma and RANTES) or non-survival (MCP-1). The role of these cytokines was investigated using recombinant virus expressing each cytokine and knockout mice of the cytokine or its receptor. MCP-1 expressing virus and MCP-1 and MCP-1 receptor knockout mice had increased pathogenicity when compared to wild type virus. The MCP-1data suggests that macrophages are essential for controlling infection, but excessive MCP-1 alter macrophage function which may exacerbate disease. IFN-gamma expressing virus did not develop disease, and knockout of IFN-gamma or its receptor resulted in increased pathogenicity. Mice inoculated with interferon gamma expressing virus did not develop any signs of disease and survived lethal challenge at the equivalent 100% lethal dose of wild type virus. Expression of interferon gamma at the site of infection via the recombinant virus resulted in protection from challenge with the LD100 of wild type virus. 2. Bivalent vaccines that confer protection against rabies and Ebola virus We have been developing a rabies virus based vaccine that expresses the glycoprotein (GP) from ebolavirus, marburgvirus and sudanvirus. Previously we developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Zaire ebolavirus (EBOV) glycoprotein (GP) using a reverse genetics system based on the SAD B19 RABV wildlife vaccine in collaboration with Matthias Schnell of Thomas Jefferson University. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced cellular and humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. We evaluated our vaccine candidates in a rhesus macaque challenge model. 100% protection was observed with live attenuated RABV-GP. The inactivated and RVdelG-GP viruses provided 50% protection. Strong humoral and cellular immunity was observed. In summary, our findings indicated that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVdelG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use. We also further developed our vaccine constructs to use codon optimized filoviruses and have demonstrated immunogenicity and safety of these constructs in mice. 3.Filovirus Molecular Virology We are also investigating molecular virology of filoviruses. We are studying the role of phosphorylation of the viral proteins in the virus lifecycle. We have chosen to focus on VP35 as it is essential for transcription and replication of the genome. Using a replicon system we have generated point mutations and clustered point mutations of potential phosphorylation sites and evaluate the mutant for replication and transcription. Our data indicates a hierarchical phosphorylation strategy that regulates VP35 function. A second project involves identification of structure and function relationships of the EBOV noncoding regions. We have identified host proteins that interact with these EBOV genome regions. We are working to identify the proteins. We have identified HSC70 as interacting with at least one domain in the EBOV Trailer by pull-down assays and confirmed using immunoprecipitation-RT-PCR. Three HSC70 binding domains have been identified within the EBOV Trailer and mutational analysis of the 5 most region results in a decrease in replicon signal. We have also undertaken SHAPE analysis of the EBOV Trailer to define the secondary structure of the EBOV Trailer. Based on these data we may identify therapeutic targets as well as establish mechanisms of filovirus lifecycle regulation. 4. MERS-CoV We have also been developing NHP models for MERS-CoV. We have established that intratracheal inoculation with MERS-CoV Jordan isolate results in limited respiratory tract disease that peaks between days 4 and 5 post inoculation, resolves by day 9 post inoculation and can be quantified by computed tomography. Inoculation of common marmosets also results in a longer lived respiratory tract disease that peaks between days 3 and 14, but does not completely resolve, and can be monitored and quantified by computed tomography. Infectious virus could not be recovered from serial samples in either rhesus or common marmosets. To further develop the MERS-CoV model we are working with Dr. Ralph Baric to construct and evaluate a MERS-CoV that expresses the human sodium-iodide symporter that can be used as a PET reporter. We expct this recombinant virus to provide enhanced detail on the infectious process of MERS in NHPs and other animal models. We have also entered a collaboration with SAB and NMRC to develop a polyclonal antibody to MERS-CoV as a human therapuetic